1. Field of the Invention
The invention is directed to improving the sensitivity of technology for the real-time quantification of bacterial endospore levels based on lanthanide dipicolinate luminescence.
2. Description of the Prior Art
An endospore (i.e., bacterial spore or bacterial endospore) is a generalized term for a type of spore that is produced within certain species of bacteria. The primary function of most endospores is to ensure survival through periods of environmental stress. Endospores are drought, heat, and starvation tolerant. They are protected by a hardened shell of protein and carbohydrates and produced by a form of binary fission in bacteria. Hence, endospores are the prime life form for free dispersal of Bacillus and Clostridium bacterial species in the environment, i.e. dispersal without being hosted by any other life form. The cost and technical difficulty of endospore production has continued to decrease over time, thereby coming with the economic and technological means of a wider segment of the world population, including radical ideological groups incapable of being deterred by possible retaliation and harbored by unofficial transnational political networks unburdened any national identification or responsibility. The need to be able to detect the presence of endospores in the field has become more urgent with this significant proliferation of biotechnologies capable of producing toxic endospores in quantity.
More specifically, certain bacteria can form endospores during times of stress or lack of food. This dormant bacterial form can survive harsh conditions such as boiling, freezing, and desiccation that readily kill vegetative bacteria. Indeed, Bacillus stearothermophilus and Bacillus subtilis spores are used to check the performance of autoclaves. Two genera of medical importance, Bacillus and Clostridium, have the ability to develop endospores. They are the causative agents of anthrax, tetanus, botulism, and gas gangrene. Other endospore-producing prokaryotes are found in several genera of soil bacteria such as Desulfotomaculum, Sporolactobacillus, and Sporosarcina. Endospore-forming bacteria are most commonly found in the soil. However, the endospores themselves exist almost everywhere, including the atmosphere, where they may ride on dust particles. The fact that endospores are so hard to destroy is the principal reason for the lengthy and elaborate sterilization procedures that are employed in hospitals, canneries, and other places where sterilization is required.
The current state of the art method requires lengthy and labor intensive methods. Two traditional methods are used to measure the concentration of endospores in a sample—colony counting and direct microscopic counting. The procedure for colony counting to determine endospore concentration consists of heat-shocking the sample to kill vegetative cells while endospores remain viable, plating a known volume of the sample with a known dilution factor onto a growth medium, and incubating the growth plates for 2 days. Finally, the resultant colonies are counted and reported as colony-forming units (CFU's).
The procedure for direct microscopic counting consists of two steps. (1) The sample is placed on a slide with an indentation of a known volume, and the glass surface of the slide is inscribed with squares of known area. (2) The bacteria in each of the several squares are counted and the average count is multiplied by an appropriate factor to yield the number of total cells per milliliter in the original suspension. Bacterial endospores can be specifically identified using microscopic staining methods. However, because the limited field of view of a microscope at high magnifications, searching for stained bacterial cells in low concentrations can take a prohibitive amount of time.
These methods suffer prohibitive difficulties with low-concentration field samples. First, endospores tend to agglomerate on particulates; attached endospores cannot be accurately counted with the traditional methods, even though they could easily represent the bulk of biomass in a field sample. Second, the traditional methods take a long time and are labor-intensive. Finally, colony-counting methods only work for cultivable bacteria, which are in the minority in field samples.
The prior art method of endospore detection was first worked out by L. E. Sacks, “Chemical Germination of Native and Cation-Exchanged Bacterial-Spores with Trifluoperazine,” Applied and Environmental Microbiology, vol. 56, pp. 1185-1187, 1990. This was later refined by Rosen, “Bacterial Spore Detection And Quantification Methods”, U.S. Pat. No. 5,876,960 (1999), which is incorporated herein by reference, has a detection limit of 105 spores/ml, which is too high for many applications to be practical. According to this art, a lanthanide such as europium or terbium is combined with a medium to be tested for endospore content. The lanthanide will react with calcium dipicolinate present in any bacterial spores in the sample medium to produce a lanthanide chelate, specifically, terbium or europium dipicolinate. The lanthanide chelate has distinctive absorbance and emission spectra that can be detected using photoluminescence testing, for example. The occurrence of emission from the sample medium upon excitation at wavelengths distinctive of the lanthanide dipicolinate chelate, thus reveals the presence of spores in the sample medium. The concentration of spores can be determined by preparing a calibration curve that relates absorbance or emission intensities to spore concentrations for test samples with known spore concentrations. The calibration curve can be used to determine the spore concentration of a sample medium using the absorbance or emission intensity for the combined lanthanide-sample medium.
The current state-of-the-art luminescence endospore quantification scheme suffers from low sensitivity, which is limited by the binding constant (i.e., the affinity of dipicolinic acid for binding the lanthanide ion). What is needed is a real-time endospore quantification methodology that is not limited by high detection thresholds. Such an invention has immediate uses in the healthcare field, food preparation/inspection markets and for monitoring biological warfare attacks.